Methods for cancer detection, diagnosis and prognosis

ABSTRACT

The present invention provides a method for diagnosing cancer, predicting a disease outcome or response to therapy in a patient sample. The method involves isolating a circulating tumor cell (CTC), for example, a viable CTC, from a sample using a parylene microfilter device comprising a membrane filter having or consisting of a parylene substrate, which has an array of holes with a predetermined shape and size; and detecting and quantifying telomerase activity in blood circulating tumor cells. The invention further provides methods of using cells live-captured in various applications.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/180,021, filed May 20, 2009, which is herein incorporated byreference in its entirety for all purpose.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

The U.S. Government has certain rights in this invention pursuant toGrant No. K08 CA126983-01 awarded by the National Cancer Institute(NCI).

BACKGROUND OF THE INVENTION

Cancer is a major health concern worldwide, accounting for millions ofdeaths and untold pain and suffering each year. The biologicheterogeneity of this disease and the vast populations afflicted posethe pivotal questions of whom to treat and with which therapies,challenges that can only be addressed through the development of moreaccurate and informative biomarkers. Peripheral blood circulating tumorcells (CTCs) recently have been detected and shown to have prognosticand predictive value in breast and prostate cancer (see, Ignatiadis, M.,et al., J Clin Oncol 2007; 25:5194-202; Pachmann, K., et al., J ClinOncol 2008 (ASCO Annual Meeting abstr 11001); Rack, B. K., et al., JClin Oncol 2008 (ASCO Annual Meeting abstr 503); Goodman, O. B., et al.,J Clin Oncol 2008 (ASCO Annual Meeting abstr 5169); Pittard, G., et al.,J Clin Oncol 2008 (ASCO Annual Meeting abstr 5072); Fizazi, K., et al.,Ann Oncol 2007; 183:518-21; and Shaffer, D. R., et al., Clin Cancer Res2007; 13:2023-9). However, these preliminary efforts have been hamperedby two significant limitations: (1) CTC isolation and (2) CTC detection.Collecting CTCs has involved a laborious process that employs multipleantibody binding and magnetic bead sorting steps, requiring expensivereagents and equipment, and ultimately yielding a relatively smallpopulation of CTCs, which may vary from sample to sample depending onthe expression pattern of cell surface markers used in this method.Currently, CTCs are isolated from blood by methods which rely onimmuno-magnetic binding of cell surface epithelial cell adhesionmolecules (EpCAMs), an expensive, labor-intense approach that is limitedto EpCAMexpressing tumors (see, Cristofanilli, M., et al., N Engl J Med2004; 351:781-91; and Nagrath. S., et al., Nature 2007; 450:1235-9). Analternative platform using a novel parylene-C pore microfilter whichtraps CTCs quickly and efficiently based on their size differential fromother blood cells (See, Zheng, S., et al., J Chromatogr A 2007;1162:154-61). However, like the EpCAMbased approach, the poremicrofilter still relied on fixation, staining, and visual enumerationof captured cells, a laborious and subjective process prone toreader/operator variability. Nevertheless, regardless of the isolationmethod, it is difficult to derive accurate diagnostic, prognostic orpredictive data from absolute numbers of CTCs because of the relativepaucity of these cells in peripheral blood.

Therefore, there is a need to develop simple yet highly sensitive andspecific cancer detection systems and methods to overcome the above andother problems. The detection systems and methods can be used as adiagnostic, prognostic or predictive assay in patients.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to novel systems and methods for cancerdetection, analysis and characterization. More specifically, theinvention is directed to a method for diagnosing a cancer or predictinga disease outcome in a patient sample. The method includes the use of aconstant low-pressure system coupled to a parylene filtration device torapidly and efficiently capture circulating tumor cells (CTC) and detectand quantify telomerase activity in blood CTCs. The method is useful asa diagnostic, prognostic and predictive technique in cancer. Forexample, the system and method are capable of cell capture from 1 ml ofwhole blood in less than 5 minutes, achieving greater than 90% captureefficiency, greater than 90% cell viability and greater than 200-foldsample enrichment.

In one aspect, the present invention provides a method for diagnosingcancer or predicting a disease outcome in a patient sample. The methodincludes, for example, isolating a circulating tumor cell from a sampleusing a parylene microfilter device comprising a membrane filter havingor consisting of a parylene substrate, which has an array of holes witha predetermined shape and size; and determining the telomerase activityof the isolated circulating tumor cell using, for example, a telomeraseactivity assay.

In another aspect, the present invention provides a method for detectingand quantifying the telomerase activity of a circulating tumor cell. Themethod includes, for example, isolating the circulating tumor cell froma sample using a parylene microfilter device comprising a membranefilter having or consisting of a parylene substrate, which has an arrayof holes with a predetermined shape and size; and determining thetelomerase activity of the isolated circulating tumor cell using aquantitative PCR telomerase activity assay.

In still another aspect, the present invention provides a method forenrichment of circulating tumor cells. The method includes passing asample containing a circulating tumor cell through a parylenemicrofilter device comprising a membrane filter having or consisting ofa parylene substrate, which has an array of holes with a predeterminedshape and dimension; and capturing said circulating tumor cell on themembrane filter, wherein the enrichment of circulating tumor cells isgreater than at least 200-1500 fold.

In a further aspect, the invention provides a system for isolating acirculating tumor cell. The system includes a parylene microfilterdevice comprising a membrane filter having or consisting of a parylenesubstrate having an array of holes with a predetermined shape anddimension; and a constant pressure delivery system coupled to theparylene microfilter device for maintaining a constant pressure.

In another aspect, the present invention provides a method for measuringmRNA expression levels of a gene in a circulating tumor cell (CTC). Themethod includes isolating a CTC from a sample using a parylenemicrofilter device comprising a membrane filter having or consisting ofa parylene substrate, which has an array of holes with a predeterminedshape and size; and measuring the mRNA expression levels of the gene inthe CTC.

In yet another aspect, the present invention provides a method forstaining a circulating tumor cell (CTC). The method includes isolating aCTC from a sample using a parylene microfilter device comprising amembrane filter having or consisting of a parylene substrate, which hasan array of holes with a predetermined shape and size; and staining theCTC with a dye or an antibody conjugated to a dye.

In yet another aspect, the present invention provides a method forpropagating a circulating tumor cell (CTC) in culture. The methodincludes isolating a CTC from a sample using a parylene microfilterdevice comprising a membrane filter having or consisting of a parylenesubstrate, which has an array of holes with a predetermined shape andsize; and propagating said CTC in culture.

In still another aspect, the present invention provides a method formeasuring telomerase activity in a single circulating tumor cell (CTC).The method includes isolating a group of CTCs from a sample using aparylene microfilter device comprising a membrane filter having orconsisting of a parylene substrate, which has an array of holes with apredetermined shape and size; obtaining individual CTCs from the groupof CTCs isolated from the sample; and determining telomerase activity ofthe CTC.

These and other objects, embodiments and advantages of the presentinvention will be more apparent to one of skill in the art from thefollowing detailed description and Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process for production of parylene microfilteraccording to an embodiment of the invention. (1) Silicon wafer assubstrate; (2) 10 μm parylene-C coating; (3) Cr/Au deposition; (4)Lithography; (5) Metal etch; (6) Reactive Ion etching (RIE) of parylene;(7) Remove metal mask; (8) Release parylene membrane.

FIG. 2 illustrates the experimental setups according to an embodiment ofthe invention. A. Constant pressure fluid delivery system. B.Cross-sectional view of the filtration setup. C. Top view of slotfilter. Each filter contains 30401 slots per 36 square millimeter. Thethickness of the parylene-C membrane filter is 10 μm.

FIG. 3 illustrates microfilter optimization and validation for capturingtumor cells. (A). Slot size and filtration pressure optimization using 1ml whole blood. Left: Comparison of capture efficiency and viabilitygenerated by filters with different slot sizes. Center: Measurement ofenrichment using filters with different slot sizes. Right: Comparison ofcapture efficiency and viability with various filtration pressures using6 μm slot filter. (B) Cancer cells captured on microfilter from 1 mlwhole blood. Shown are bright-field (left) and merged fluorescence(center) micrographs taken of the same field; yellow arrows (310)indicate live captured cancer cells, red arrows (320) indicate deadcancer cells, and black arrows (330) indicate PBMCs. Right: SEMmicrograph of captured cancer cell. (C) Validation of cancer cellcapture from a standard volume 7.5 ml specimen. Shown are captureefficiency (left), cell viability (middle) and enrichment (right). (D)On-filter (top) and off-filter (bottom) cell culture of captured PC-3cells from human blood after 3 days and 6 days in RPMI complete medium.

FIG. 4 shows an image of captured pre-stained tumor cells on filterspiked into blood (20×).

FIG. 5 illustrates a strategy for quantitative PCR detection oftelomerase activity according to one embodiment of the invention.

FIG. 6 illustrates qPCR-based telomere repeat amplification protocol(qPCR-TRAP). A. Standard dilution curves comparing the log of cellnumbers (from which telomerase-containing protein lysates are extracted)to the resulting qPCR threshold cycle (Ct, the amplification cycle atwhich the telomeric products made by telomerase are detected). The moretelomerase activity, the fewer qPCR amplification cycles required fordetection of products (hence lower Ct). The cell line is LNCaP; cellnumbers and the corresponding Ct's are indicated in the table. B.Histogram of qPCR-based telomerase activity of preparations consistingof various ratios of LnCaP cells and peripheral blood mononuclear cells(PBMCs); cancer cell telomerase activity is detectable above the PBMCbackground at a ratio of 1:1000.

FIG. 7 illustrates qPCR-based detection of telomerase activity from livecancer cells captured on slot microfilter. (A) Telomerase activitydetected from 7.5 ml blood samples spiked with a range of cancer cellnumbers or blood only (p=0.01 for each sample compared with blood-onlysample). (B) Linear correlation of Ct values with the number of spikedcells. Results in all histograms are means of triplicate independentexperiments. (C) Telomerase activity of patient samples versus healthydonor controls. The line in healthy donors indicates the calculatednormal cut-off value of Ct at 33; patient samples falling withinpositive range (Ct<cut-off value) are boxed. (D) Serial filtration tointernally control for PBMC background telomerase activity on 6 positivepatient samples (p=0.029) (E) Serial filtration to internally controlfor PBMC background telomerase activity on healthy donor samples(p=0.5).

FIG. 8 illustrates telomerase activity measurement from single livecancer cells captured on microfilter. (A) Captured cells stained byPE-CD49b. (B) Matched bright field image. (C) Micropipette recovery ofsingle cell. (D) Single cell telomerase activity assays.

FIG. 9 illustrates PCR for measuring transcript levels for genes ofinterest from cells live-captured on slot filter. Bothquantitative/real-time PCR (A) and standard PCR (B) were performed forPSA transcript detection and measurement.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “parylene” refers to a polymer having formulaeI, II, and III or combinations thereof. The polymer can be ahomopolymer, a copolymer, a polymer blend or combinations thereof. R¹,R², R⁷ and R⁸ are each independently H, alkyl, heteroalkyl, aryl orhalogen. The alkyl can be a C₁-C₆ hydrocarbon radical. The halogen isCl, F, Br, or I. Heteroalkyl is an alkyl substituent containing at leastone heteroatom, such as O, S, N, Si or P.

R³-R⁶ are each independently H, alkyl, aryl, halogen, heteroalkyl,hydroxyl, amino, alkylamino, arylamino, aroylamino, carbamoylamino,aryloxy, acyl, thio, alkylthio, cyano, alkoxy. An alkyl group can be asubstituted alkyl having up to 29 carbon atoms. A substituted alkyl canbe mono- or polyunsaturated alkenyl or alkynyl radical having in eachcase up to 29 carbon atoms, i.e., a substituted C₁-C₂₉alkyl,C₂-C₂₉alkenyl or C₂-C₂₉alkynyl radical. Suitable substitutents are alsocyclic radicals. The substituted alkyls can be methyl, ethyl, or propylradical, carrying one or more identical or different radicals. Dependingon the nature of the substitutents, these can be attached via a singleor multiple bond or in a spiro form. Preferred substitutents arehalogen, such as Cl, F, Br or I, amino, lower alkylamino, loweralkanoylamino, aroylamino, such as, in particular, benzoyl amino,hyroxyamino, hydroxyimino, lower alkoxyamino, aroxyamino, such as, inparticular, phenoxyamino. Lower alkylthio includes C₁-C₆alkylthiols.Aryloxycarbonyl includes phenoxycarbonyl, benzyloxycarbonyl,hydroxyaminocarbonyl, aminoacylamino, carbamoyl, amidino. Aryoxy can bephenyloxy, aminocarbonyl-oxy, oxo, aminosulfonyl and loweralkylsulfonyl-amino. Heteroalkyl is an alkyl substitutent having one ormore heteroatoms in the alkyl substitutents, in particular,mercaptoalkyl having up to 29 carbon atoms, aminoalkyl, phosphinoalkyl,haloalkyl, hydroxyalkyl or silylalkyl. Preferably, parylene has astructure represented by the formula I. In addition, preferred parylenealso includes commercially available parylene, C, F, A, AM, N, and D.

As used herein, “polymerase” refers to an enzyme that catalyzes thepolymerization of nucleotides (i.e., the polymerase activity).Generally, the enzyme will initiate synthesis at the 3′-end of theprimer annealed to a polynucleotide template sequence, and will proceedtoward the 5′ end of the template strand.

As used herein, the term “primer” refers to a single stranded DNA or RNAmolecule that can hybridize to a polynucleotide template and primeenzymatic synthesis of a second polynucleotide strand. A primer usefulaccording to the invention is between 10 to 100 nucleotides in length,preferably 17-50 nucleotides in length and more preferably 17-45nucleotides in length.

As used herein, “Quantitative PCR” or “QPCR” is defined as a polymerasechain reaction (PCR) process which monitors the kinetics of PCR for thequantification of DNA templates. When QPCR follows a reversetranscription reaction, it can be used for the quantification of RNAtemplates as well. The amplification of nucleic acid using qPCR is atransformation of a single strand of DNA into multiple copies.

As used herein, the term “monodispersed” refers to openings or holes onthe membrane filter having substantially identical size, dimension andshape.

As used herein, the term “prognosis” defines a forecast as to theprobable outcome of a disease, the prospect as to recovery from adisease, or the potential recurrence of a disease as indicated by thenature and symptoms of the case.

As used herein, the term “figure of merit” provides a measure of theefficiency of the filtration device. A large figure of merit number isan indication of higher filtration efficiency. Figure of merit isdefined as the recovery rate divided by time. Recovery rate is definedas particles recovered divided by the total number of target particles.The time used in the calculation of figure of merit is the totalprocessing time to conduct the testing. For example, in one embodiment,the parylene filter of the present invention has a figure of merit ofgreater than or equal to 890 and/or having a Young's modulus ≈4 GPa.

Parylene is a USP Class VI biocompatible polymer that can be depositedthrough a highly-conformal vapor deposition process. Types of paryleneinclude parylene C, F, A, AM, N, and D. Of the three most common typesof parylene shown below, parylene C is perhaps the most widely used inindustry. The advantages of the use of parylene include its provenbiocompatibility, its strength, elasticity and flexibility (e.g.,Young's modulus ≈4 GPa), its conformal pinhole-free room-temperaturedeposition, its low dielectric constant (≈3) high volume resistivity(>10¹⁶ Ω-cm), its transparency, and its ease of manipulation usingstandard microfabrication techniques such as reactive ion etching (RIE).In certain embodiments, the parylenes used in the present invention haveYoung's modulus of at least 4 GPa. Several research groups have usedparylene C deposition as a method of creating a biocompatible,water-blocking seal around electrode arrays typically fabricated using apolyimide substrate. This is necessary because most polyimides have amoisture absorption that is more than an order of magnitude higher thanthat of parylene C. Some specialized polyimide films have lower moistureabsorption, but they require high-temperature curing steps.

Parylene microfilters have found various applications. Parylenemicrofilter devices with predetermined geometric design have beendescribed in U.S. Patent Publication No. 2006/0254972 as incorporatedherein by reference. Use of parylene microfilters for various biologicalapplications has been described in U.S. Patent Publication No.2007/0025883 and PCT Patent Publication No. WO2006/116327, each of whichis incorporated herein by reference. Parylene microfilters with a topand a bottom porous membranes for separating circulating tumor cellshave been described in U.S. Patent Publication No. 2009/0188864, whichis incorporated herein by reference.

In one aspect, the present invention provides a highly sensitive yetspecific method for diagnosing cancer and/or predicting disease outcomeand response to therapy by (i) isolating circulating tumor cells frombody fluids, such as blood samples including peripheral blood samplesusing a parylene microfilter device, such as a parylene-C slotmicrofilter device, and (ii) determining the telomerase activity of thecells using a telomerase assay, such as a quantitative PCR-baseddetection. The parylene microfilter device includes a membrane filterhaving or consisting of a parylene substrate, which has an array ofholes with a predetermined shape and size. In one embodiment, the methodincludes correlating the telomerase activity with malignant ormetastatic potential. Advantageously, the captured cells retained normalmorphology by scanning electron microscopy and can be readilymanipulated, further analyzed, or expanded on or off filter. Remarkably,telomerase activity, a well-recognized universal cancer marker, isreliably detected by qPCR from as few as 25 cancer cells spiked into 7.5ml whole blood and captured on microfilter. Moreover, significanttelomerase activity elevation also was measured from patient bloodsamples, and even from single cancer cells lifted off the microfilter.Live CTC capture and analysis is fast and simple yet highlyquantitative, versatile, and applicable to nearly all solid tumor types,making this suitable for cancer detection and characterization.

The parylene membrane filter comprises a plurality of holes of apredetermined geometric design formed in, and penetrating, the parylenemembrane. The geometric design includes, for example, a size, a shapeand density. In one embodiment, the design of the membrane is such thatCTCs are selectively captured or retained by the membrane while othercells and materials in the blood pass through the membrane selectedaccording to their size and shape. The efficiency of the membrane filtercan be adjusted by changing the size, shape, density of the holes on themembrane and the pressure applied to the sample to be filtered. In somepreferred embodiments, the filter of the present invention has a figureof merit up to 890. In other embodiments, the parylene membrane filterhas a figure of merit between about 800 to about 890. In some preferredembodiments, the holes are monodispersed.

The predetermined geometric design is according to any one or more ofthe size, shape, density, uniformity, and arrangement of the holes inthe parylene membrane. In some embodiments, the holes themselves canhave rounded or sharp corners. The holes can be of a regular shape(e.g., circles, ovals, ellipses, squares, rectangles, symmetrical andunsymmetrical polygons, rods) or any other shape desired, including, butnot limiting to, other irregular shapes. The holes can be of differentsizes and shapes. The holes can all be of uniform size and/or shape. Insome preferred embodiments, the holes may be limited to a predeterminedrange of sizes and/or shapes. In some embodiments, membrane filter has ahole shape selected from the group consisting of a circular, anelliptical, a symmetrical polygonal, an unsymmetrical polygonal, anirregular shape and combinations thereof. In a preferred embodiment, theholes have a rectangular shape and arranged uniformly. In someembodiments, the holes can be arranged in a uniform grid or array (e.g.,one or more rows and/or columns, concentric circles, and the like).Preferably, holes are all of the same shape and size and may also be ofuniform density or pattern on the membrane, aside from the edges.

The holes can be of any desirable size and shape which will determinethe ability of a particle or cell of interest to pass through. Forinstance, in some embodiments, the holes may have a minimum or maximumcross sectional length of 1, 2, 3, 4, 5, 8, 10, 12, 14, 16, 18, 20, 24,28, 30, 32, 36, 40, 45, 50 microns or more. In some embodiments, theholes are circles, ovals, rectangles or polygons. In some furtherembodiments, the circular holes have diameters of 2, 4, 8, 10, 14, 20,30, 40, 50 microns or more. In other further embodiments, the holes areoval and have different lengths and widths which may be independently beselected from 2, 4, 6, 8, 10, 14, 20, 30, 40 or 50 microns. Forinstance, in some further embodiments, the holes may be circles from 6to 10, 5 to 12, 10 to 20, 8 to 40, or 6 to 60 microns in diameter. Insome preferred embodiments, the holes are rectangles whose dimensionsare from 2 to 10 microns by 30 to 60 microns, from 4 to 9 microns by 35to 50 microns, from 5 to 8 microns by 35 to 45 microns, or from 5 to 7microns by 35 to 45 microns. In a more preferred embodiment, the holesare from 6 by 40 microns. In some embodiments, the minimum width of therectangular holes is 1, 2, 4, 5, 5.5, 6, 6.5, 7, 8, 9 or 10 microns andthe minimal length of the rectangular holes is 30, 31, 32, 33, 34, 36,38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50 microns. In some embodiments,the width of the rectangular holes is 1, 2, 4, 5, 5.5, 6, 6.5, 7, 8, 9or 10 microns and the length of the rectangular holes is 30, 31, 32, 33,34, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50 microns. In someembodiments, of any of the above the maximum length of the hole is 50,60, 70, 80, 90, 100 microns or 200 microns.

The holes may also be defined according to their cross sectional areaand/or shape. The shapes can be as any described above, preferably, theshapes are rectangle. In some embodiments, the cross sectional areasrange from about 1 to 1000 square microns, 1 to 10 square microns, 10 to100 square microns, 25 to 500 square microns, 50 to 400 square microns,75 to 150 square microns, 75 to about 500 square microns or 200 to 1000square microns. In certain embodiments, the cross sectional areas rangefrom 50 to 300 square microns, 100 to 200 square microns, 200 to 240square microns, 150 to 300 square microns, 200 to 280 square microns or200 to 400 square microns. In one embodiment, the holes have aslot/rectangular shape and a cross sectional area of 240 square microns.In any of the above, the holes can be monodispersed. In any of theabove, the parylene membrane filter can have a figure of merit up to890, and preferably from 800 to 890.

In some embodiments, the parylene membrane filter has a hole density offrom 1 to 40,000, 1,000 to 40,000, 5,000 to 40,000; 6,000 to 40,000,7000 to 40,000, 10,000 to 40,000; 10,000 to 30,000; 20,000 to 30,000;20,000 to 40,000; or 30,000 to 40,000 holes per square millimeter. Incertain instances, the parylene membrane filter has an array ofrectangle holes with a hole density from 1 to 1000, 1 to 900, 1 to 850,1 to 800, 1 to 700, 1 to 600, 100 to 1000, 300 to 1000, 500 to 900, 400to 800, or 600 to 900 holes per square millimeter. In one instance, theparylene membrane filter has an array of rectangle holes with a holedensity of 100, 200, 300, 400, 500, 600, 700, 800, 850, 900 or 1000holes per square millimeter. In certain embodiments, the hole density isat least 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000 or 8000 holes per square millimeter. In apreferred embodiment, the parylene membrane filter has 30401 or moreholes per 36 square millimeters. Such hole densities depend in part uponthe sizes of the holes, with smaller holes allowing for greaterdensities. The densities can be adjusted so as to insure that the holesdo not fuse together during manufacture and the strength of the parylenemembrane remains suitable. A thicker membrane can be used to strengthenthe membrane at higher hole densities.

In certain embodiments, the number and size of the holes affects therate at which a sample can pass through the membrane and the strength ofthe membrane. The density of the holes is typically range from 1,000 to40,000 holes per square millimeter. The plurality of holes can providean opening area ratio of from 4% to 60%, including ranges from 4% to25%, 5% to 25%, 10% to 25%, 15% to 30%, 5% to 45%, 10% to 50%, 15% to45%, 20% to 40%, 25% to 50%, and 45% to 60%. In some embodiments, thearea opening ratio is at least 1%, 2%, 4%, 5%, 8%, 10%, 12%, 13%, 14%,15%, 17%, 18%, 19% or 20%. In one embodiment, the area opening ratio is18%.

In some embodiments of the above, the parylene membrane filter is from0.5 to 20 microns thick. In some preferred embodiments, the membrane isat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 micronthick. In other embodiments, the membrane filter is from 1 to 20 micronsthick, in more preferred embodiments, the membrane filter is from 1 to4, 5 to 10, 5 to 15, 8 to 15 or 10 to 20 microns thick. In oneembodiment, the parylene membrane filter has a thickness of 10 microns.The thickness of the membrane filter is a compromise between membranestrength and flow resistance through the membrane. Accordingly, asincreasing hole density reduces membrane strength, membranes having agreater number of holes typically require a thicker membrane thanmembranes having a fewer number of the same holes.

In some embodiments, a constant pressure can be applied to the sample tofacilitate the filtration process, preferably, a constant low-pressureis applied to the sample. The pressure can range from 0.01 to 0.5 psi,preferably from 0.05 to 0.4 psi, more preferably from 0.1 to 0.3 psi andeven more preferably from 0.1 to 0.25 psi. In one embodiment, theconstant pressure is 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17,0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25 psi. In anotherembodiment, a maximum constant pressure applied to the sample is 0.1,0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22,0.23, 0.24, 0.25, 0.36, 0.27, 0.28, 0.29 or 0.3 psi. In one embodiment,the pressure applied to the sample is generated by an electrokinetic,for example, electroosmosis, and a ratchet pump. In yet anotherembodiment, the pressure is generated using pneumatic or magnetohydrodynamic pumps. In yet a further embodiment, the pressure applied tothe fluid is generated by a mechanical device. One example of a usefulmechanical pressure generating device is a screw-type pumping device ora peristaltic pump. In a preferred embodiment, the pressure is generatedthrough a compress gas source. Exemplified gases include nitrogen,argon, helium, air, carbon dioxide or oxygen. The pressure is preferablyapplied through a pressure delivery system, for example, as depicted inFIG. 2A.

The filtration sample can be any body fluid containing tumor cells. Forinstance, the sample can be a blood sample from a mammal. In oneembodiment, the sample is a peripheral blood sample obtained from apatient.

In some embodiments, the invention provides a method for determining theisolated CTCs using a telomerase activity, such as determined by anassay. The method further provides correlating the telomerase activitywith malignant or metastatic potential. Telomerase is a specializedreverse transcriptase that through a transformation synthesizestelomeric DNA and thus contributes to the maintenance of functionaltelomeres. In most cancers and in stem cells of renewal tissues,telomerase activity levels generally correlate with the proliferationstate of the cells. The presence of the enzyme is almost always requiredfor unlimited proliferation (immorality) whereas its absence may dictatea finite lifespan (senescence). In fact, telomerase activity is awell-recognized cancer marker in >90% of human malignancies (Streutker,C. J., et al., Pediatr Dev Pathol 2001; 4:62-7) and therefore is ideallysuited to the microfilter, which can capture CTCs across all tumor typesregardless of surface markers. Telomerase activity also constitutes auniquely “functional” assay which reflects the presence of live cancercells, in contrast to other CTC readouts such as cancer-specific geneproducts or mutations that can be falsely amplified by qPCR from deadcells or cellular debris. Moreover, qPCR-TRAP can amplify the telomeraseactivity signal through a transformation from as few as one cancer cell,raising the prospect of applying CTC-telomerase not only as a prognosticor predictive biomarker, but also as a method for early screening anddetection of occult malignancy. The versatility and wide applicabilityof CTC live-capture and analysis make this a powerful new strategy forthe study of cancer dissemination and for the advancement of patientcare.

It is known in the art that tumor samples can be assayed by PCR-basedtelomeric repeat amplification protocol (TRAP) (Holt, S. E., et al.Methods Cell Sci 1996, 18, 237-248). TRAP is a two-step process. A cellor tissue sample is lysed with a buffer containing detergent, and analiquot of lysate is mixed with a reaction solution containing elementsfor the two-step process of telomerase product formation andamplification. In the first step, the telomerase substrate and dNTPswithin the reaction solution are used for the addition of telomericrepeats by telomerase if it is present within the sample lysate(represented as a ladder on acrylamide gels). This step involves atransformation. In the second step, forward and reverse primers forthese products are used for amplification. The amplification verifies atransformation. Real time quantitative TRAP allows a more rapid,high-throughput, quantitative analysis of telomerase activity in cell ortissue samples; therefore, this assay is optimal for clinical use(Elmore, L. W. et al. Diagn. Mol. Pathol. 11, 177-85, (2002); andJakupciak, J. P. Expert Rev. Mol. Diagn. 5, 745-53 (2005)). Analysis ofQ-TRAP described in this protocol is based on standard real-time PCRanalysis, which uses a relative standard curve method. The cyclethreshold (Ct) of an unknown sample is compared to a standard curve toquantify the relative amount of telomerase activity, which can then benormalized to the standard.

In one embodiment, the determination of the telomerase activity includesobtaining a telomerase extension product; and amplifying the extensionproduct by a quantitative PCR-based telomerase repeat amplificationprotocol assay. In one instance, the telomerase extension product isobtained by lysing the isolated circulating tumor cell to produce a celllysate; and mixing the cell lysate with an oligonucleotide that is asubstrate for telomerase extension to obtain an extension product.

In another aspect, the present invention provides a method for detectingand quantifying the telomerase activity of a circulating tumor cell. Themethod includes isolating the circulating tumor cell from a sample usinga parylene microfilter device comprising a membrane filter consisting ofa parylene substrate having an array of holes with predetermineddimensions and sizes; and determining the telomerase activity of theisolated circulating tumor cell using a quantitative PCR telomeraseactivity assay. In one embodiment, the sample is a peripheral bloodsample. Preferably, the isolated cells are alive. In some embodiments ofany of the above, the holes have a rectangular shape. The width of therectangular holes is from 1 to 10 microns, preferably 2 to 8 microns,more preferably 4 to 7.5 microns and even more preferably 5 to 7microns; and the length of the rectangular holes is from 30 to 50microns, preferably 35 to 45 microns, more preferably 37 to 42 micronsand even more preferably 38.5 to 41.5 microns. In one embodiment of anyof the above, the rectangular hole size is 5.5 to 6 by 39.5 to 40microns. In one instance the rectangular hole size is 5.5 by 40 micronsor 6 by 40 microns. In some embodiments, the rectangular holes have awidth of 1, 2, 4, 5, 5.5, 6, 6.5, 7, 8, 9 or 10 microns and a length of30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47.48, 50 microns. In other embodiments, the rectangular holes have aminimum width of 1, 2, 4, 5, 5.5, 6, 6.5, 7, 8, 9 or 10 microns and aminimum length of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47. 48, 50 microns. In yet other embodiments, therectangular holes have a maximum width of 1, 2, 4, 5, 5.5, 6, 6.5, 7, 8,9 or 10 microns and a maximum length of 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48, 50 microns. In someembodiments of any of the above, the sample is passed through theparylene filter under a constant-low-pressure. The pressure can rangefrom 0.01 to 0.5 psi, preferably from 0.05 to 0.4 psi, more preferablyfrom 0.1 to 0.3 psi and even more preferably from 0.1 to 0.25 psi. Inone embodiment, the constant pressure is 0.1, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25 psi.In another embodiment, a maximum constant pressure applied to the sampleis 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21,0.22, 0.23, 0.24, 0.25, 0.36, 0.27, 0.28, 0.29 or 0.3 psi.

In yet another aspect, the present invention provides a method forenriching circulating tumor cells. The method includes passing a samplecontaining a circulating tumor cell through a parylene microfilterdevice comprising a membrane filter consisting of a parylene substratehaving an array of holes with a predetermined shape and size; andcapturing said circulating tumor cell on the membrane filter, whereinthe enrichment of circulating tumor cells is greater than 200, 300, 400,500, 600, 700, 900, 1000, 1100, 1200, 1300, 1400, 1500 or 2000 fold. Inone embodiment, the sample is filtered under a constant-low-pressure. Inanother embodiment, the method provides a greater than 90% captureefficiency and greater than 90% cell viability. In some preferredembodiments, the method provides a greater than 91, 92, 93, 94, 95, 96,97, 98 or 99% capture efficiency. In other preferred embodiments, themethod provides a greater than 91, 92, 93, 94, 95, 96, 97, 98 or 99%cell viability. The sample, the parylene filter, the shape and size ofthe array of holes and the pressure are as defined in any of theembodiments above. In a preferred embodiment within any of the aboveembodiments, the holes have a rectangular shape with the dimension asdescribed in any of the above embodiments.

In still another aspect, the present invention provides a system forisolating a circulating tumor cell. The system includes a parylenemicrofilter device comprising a membrane filter consisting of a parylenesubstrate having an array of holes with a predetermined shape anddimension; and a constant pressure delivery system coupled to theparylene microfilter device for maintaining a constant pressure. Thesample, the parylene filter, the shape and size of the array of holesand the pressure are as defined in any of the embodiments above. In oneembodiment, the pressure is between 0.01 and 0.3 psi. In a preferredembodiment, the holes have a rectangular shape with the dimension asdescribed in any of the above embodiments.

In another aspect, the present invention provides a method for measuringmRNA expression levels of a gene of interest in a circulating tumor cell(CTC). The method includes isolating a CTC from a sample using aparylene microfilter device comprising a membrane filter consisting of aparylene substrate having an array of holes with a predetermined shapeand size; and measuring the mRNA expression levels of the gene in theCTC. In some embodiments, the mRNA expressions, tumor markers, areproteins including, but not limiting to, Alpha-fetoprotein (AFP),Beta-2-microglobulin (B2M), Beta-HCG, Bladder tumor antigen (BTA), CA15-3, CA 27.29, CA 125, CA 72-4, CA 19-9, Calcitonin, Carcinoembryonicantigen (CEA), Chromogranin A, Epidermal growth factor receptor (EGFR),Hormone receptors, HER2 (also known as HER2/neu, erbB-2, or EGFR2),Human chorionic gonadotropin (HCG), Immunoglobulins, Neuron-specificenolase (NSE), NMP22, Prostate-specific antigen (PSA), Prostatic acidphosphatase (PAP), Prostate-specific membrane antigen (PSMA), S-100,TA-90 and Thyroglobulin. Examples of cancers linked to the above markersinclude, but are not limited to, bladder cancer, breast cancer,colorectal cancer, gestational trophoblastic disease, liver cancer, lungcancer, melanoma skin cancer, multiple myeloma, ovarian cancer,pancreatic cancer, prostate cancer, stomach (gastric) cancer, testicularcancer, seminoma and non-seminoma. Examples of oncogenes found in tumorsinclude, but are not limited to, BRCA1, BRCA2, RAS (also called HRAS),BCR-ABL, MYC, MSH2, MSH6, MLH1, CDKN2, HPC1, erb-B2, PI3-K, AKT andβ-catenin.

In another aspect, the present invention provides a method for staininga circulating tumor cell (CTC). The method includes isolating a CTC froma sample using a parylene microfilter device comprising a membranefilter consisting of a parylene substrate having an array of holes witha predetermined shape and size; and staining the CTC with a dye or anantibody conjugated to a dye. Various dyes and antibodies conjugated toa dye known in the art can be used for staining the CTCs. Exemplary dyesinclude, but are not limited to, Acridine orange, Bismarck brown,Carmine, Coomassie blue, Crystal violet, DAPI, Eosin, Ethidium bromide,Acid fuchsine, Haematoxylin, Hoechst stains, Iodine, Malachite green,Methyl green, Methylene blue, Neutral red, Nile blue, Nile red, Osmiumtetroxide (formal name: osmium tetraoxide), Rhodamine and Safranin.

The invention also provides a method of expanding or propagating afilter-captured cell in culture. The method includes isolating a CTC,such as a viable CTC from a sample using a parylene microfilter device,which comprises a membrane filter consisting of a parylene substratehaving an array of holes with a predetermined dimension and size; andpropagating the cell in culture either on filter or by washing them intoa culture dish. The conditions for culturing a cell are well known topersons of skill in the art. For example, the cells can be maintained inan incubator at 5% CO₂ and 37° C.

In yet another aspect, the present invention provides a method formeasuring telomerase activity in a single circulating tumor cell (CTC).The method includes isolating a group of CTCs from a sample using aparylene microfilter device comprising a membrane filter consisting of aparylene substrate having an array of holes with a predetermined shapeand size; obtaining individual CTCs from the group of CTCs isolated fromthe sample; and determining telomerase activity of the CTC. In oneembodiment, a micropipette is used to recover individual CTCs from agroup of CTCs isolated from a sample.

FIG. 1 illustrates a parylene-C microfilter membrane fabricationprocess. The filter fabrication process started from depositing, forexample, a 10 μm-thick parylene-C layer on prime silicon wafer.Parylene-C layer with other thickness as described above can also beused. A metal layer, either Cr/Au or Al, was then deposited using athermal evaporator, followed by lithography and wet-etching patterning.Parylene-C was patterned by Reactive Ion Etching (RIE) using the metalas the mask. Finally, the patterned parylene-C membrane was peeled offfrom the silicon substrate.

FIG. 2 illustrates a parylene filter device system 200 for capturingviable CTCs. The microfilter device is capable of enriching and trappingCTCs with unprecedented efficiency based on the size and deformabilitydifferences between CTC and blood cells. Size based filtration method isan efficient way to enrich CTCs (15-30 μm in diameter) from RBCs (6-9μm) and WBCs (5-16 μm). Advantageously, the slot size is 5.5×40 μm,which allows most RBCs and WBCs to deform and pass through, but cansuccessfully trap CTCs. The parylene filter device system consists of aconstant-pressure driven fluid delivery system coupled to a parylenefilter assembly 205 (FIG. 2B). The filter device includes a parylenemembrane 250 mounted inside a housing. The housing can adopt a varietyof sizes and shapes, which include, but is not limited to, tubular,spherical and cubical shapes. In one embodiment, the housing is made ofa top chamber 272 having an insertion port connected to a tubing 230 anda bottom chamber 274 having an exit port 260. Various materials can beused for the construction of the chambers. The materials include, butare not limited to, polysiloxane, polycarbonate, polyacrylate,polyethylene, polystyrene, polysaccharides and copolymers andcombinations thereof. In one embodiment, the material used forconstruction of the chambers is polydimethylsiloxane (PDMS). In oneembodiment, the top chamber, the bottom chamber and a parylene membraneare clamped together by two pairs of clamps 210 a and 210 b. The clampscan be made of polyacrylate, polyketone, polystyrene, polypropylene andthe like or an engineering material, which includes, but is not limitedto, a polyketone, a polysulfone, a polysulfide, a polyimide or apolyetheretherketone (PEEK). The clamps are held together by suitablemeans, such as bolts, fasteners, screws, latches, links, joints, locksor unions. The parylene membrane filter 250 has an array of rectangular(slot) holes. Compared to the circular pores, slots allow easierdeformation of blood cells in the slot longitudinal direction, whichfacilitates easier passage of normal blood cells. In addition to theslot design, the large fill factor also greatly reduces the flowresistance during filtration.

In FIG. 2A, the constant-pressure driven fluid delivery system includesa nitrogen tank, a pressure regulator, a pressure gauge, a fine pressurecontrol needle valve 280, switches and sample. The fine pressure controlvalve is capable of driving the sample with an accuracy of ±0.01 psi. Inprevious filtration approaches, samples were driven by hand-push,syringe pump or other means. Although a near-constant flow rate could beachieved, the pressure drop (ΔP) across the filter increased when thefilter was gradually clogged. Advantageously, theconstant-pressure-driven fluid delivery system is able to keep aconstant ΔP during filtration. Not being bound by any theory, the slotfilter's reduced flow resistance then allows a low and constant ΔP tofilter out CTCs, which minimizes the forces exerted on the cells, andhence results in high viability. Surprisingly, under aconstant-low-pressure driven system, the parylene filters with arectangular hole shape and predetermined hole size have been found toprovide high CTC capture efficiency (>90%), high cell viability (>90%)and high CTC enrichment (>200 fold).

FIG. 3A shows the capture efficiency, viability and enrichment of CTCsusing parylene filters having sever different slot sizes. Preferred slotwidth is about 6 microns for prostate cancer cells. High pressuresreduce capture efficiency and cell viability. Low pressure is criticalfor live cell capture. Preferred drive pressure is about 0.13 psi. Thelow pressure preserves the morphology of captured cells (FIG. 3B) andallows fast filtration. For example, filtration of 1 mL whole bloodusing the system of the present invention is done in less than 5minutes, a capture rate that is 12 times faster than that of otherrecently-published microfluidic platforms (Nagrath, S. et al., Nature2007; 450:1235-9). Using the filtration system of the present invention,a 1500 fold enrichment of CTCs is observed (FIG. 3C). In someembodiments, the filtration system of the present invention providesenrichment of CTCs for at least 200, 300, 400, 500, 600, 800, 1000,1200, 1500, 1600, 1800 or 2000 fold.

As shown in FIG. 3D, the captured cancer cells can be expanded inculture either directly on filter or by first washing them into aculture dish (FIG. 2D). Notably, the parylene microfilter provides abiocompatible environment for cancer cell adherence and growth oralternatively could release the captured cells without damaging them.These properties would allow potential expansion and study of capturedCTCs for cancer phenotyping and treatment selection. The parylene slotfilters have the advantages of biocompatibility and capability ofrelease captured cells. FIG. 3D shows that for on-filter culture, evenwithout any surface pre-treatment, cancer cells are able to adhere andproliferate on the filter surface. For off-filter culture, trapped cellsare first released and collected from the filter, by multiple rinses orreverse flow (passing PBS from backside), and then cultured inpetri-dish.

FIG. 4 shows that cancer cells can be successfully isolated from bloodusing the parylene-C based slot microfilter. For example, prostate tumorcells DU145 were pre-stained with carboxyfluorescein succinimidyl ester(CFSE). Tumor cells were then counted and spiked into blood. The buffycoat layer (containing nucleated cells) was extracted by Ficoll gradientcentrifugation, and these cells were filtrated by the slot filter usingconstant pressure driving system. FIG. 4 shows captured tumor cells onfilter. Tumor cells were still alive and fluorescence dye was keptinside the intact membrane (see also FIGS. 3B and 3C).

Compared to normal cells, most cancer cells have higher telomeraseactivity, which directly indicates their malignant/metastatic potential(see, Fizazi, K. et al., Annals of Oncology, vol. 18, pp. 518-521, 2007;and Herbert, B. S. et al., Nature Protocols, vol. 1, pp. 1583-1590,2006). Moreover, most cancer cells possess very high telomerase activitythat correlates directly with malignant/metastatic potential (see, Shay,J. W. et al. Eur. J. Cancer 1997, 33,787-791; and Meeker, A. K. et al.Urol One. 2006, 24, 122-130), a property which has demonstratedprognostic utility when measured in body fluids and tumor specimens(see, Carey, L. A. et al., J Clin Oncol 1999, 17, 3075-81; Oishi, T. etal., Obstet Gynecol 1998, 91, 568-71; Poremba, C. et al., J Pathol 2002,198, 181-89; Tomoda, R. et al., Cancer 2002, 95, 1127-33; and Sanchini,M. A. et al., JAMA 2005, 294, 2052-56). Hence, the measurement oftelomerase activity of CTCs is important for cancer metastasis study.Telomerase activity is preferably measured from viable cells. Althoughthe telomerase activity of individual whole blood cell (WBC) is low,without an efficient enrichment, the telomerase activity from all theWBCs may still add high background noise to the telomerase activity ofCTCs. The parylene filtration system of the present invention providesenrichment of CTCs and allows picking up a single cancer cell usingmicropipette from the enriched viable cancer cells on the filter andisolating the single cancer cell and obtaining its telomerase activityby sensitive quantitative PCR (qPCR). qPCR-based detection of telomeraseactivity involves a two-step assay: (1) The microfilter-trapped CTCs arelysed, and the cell lysate (which contains telomerase) is mixed with anoligonucleotide that serves as a substrate for telomerase extension, theextension product is a result of a transformation; and (2) The extensionproduct is then amplified by quantitative PCR (see, FIG. 5 and Herbert,B. S. et al., Nat Protoc 2006, 1, 1583-90).

Experiments were performed using cancer cells introduced intofreshly-drawn whole blood and showed that telomerase signal could bereliably obtained and correlated to the number of cancer cellsintroduced (FIGS. 7A and 7B). FIG. 7A shows a linear correlation betweenthe number of cancer cells seeded and resulting telomerase activity.FIG. 7B shows the PCR threshold cycle value (Ct) value is inverselyproportional and linearly correlated with the number of spiked cells. Inone embodiment, pre-determined numbers of cancer cells are “spiked” intofreshly collected 7.5 ml blood samples from healthy volunteers. Theblood containing the cancer cells are Ficoll centrifuged to separate the“buffy coat” (containing nucleated cells) from red blood cells, and thebuffy coat contents is passed through the parylene-C slot microfilter toseparate and trap the cancer cells from white blood cells. Themicrofilter trapped cells are lysed, and the cell lysates are subjectedto qPCR telomerase detection. The CTCs that can be captured and detectedby the methods of the present invention include, but are not limited to,prostate cancer cells, lung cancer cells, breast cancer cells,colorectal cancer cells, bladder cancer cells, endometrial cancer cells,kidney cancer cells, leukemia, melanoma, skin cancer cells, thyroidcancer cells, non-hodgkin lymphoma, pancreatic cancer cells andhepatocellular carcinoma. This is surprising in view of the fact thatthat hepatocellular carcinoma cannot be assayed using EpCAMs.

As shown in FIGS. 7D and 7E, when the CTC telomerase platform wasapplied to clinical samples from men with metastatic prostate cancerversus healthy controls, 5 of 12 patient blood samples had significanttelomerase activity elevation, with an average Ct of 31.4. In contrast,0 of 6 healthy donor samples had telomerase activity elevation, with anaverage Ct of 33.4; hence, the positive patient samples possessedapproximately 4-fold telomerase activity relative to the healthy donorsamples.

The telomerase activity assay can be controlled to reflect only thecaptured CTCs and not the background peripheral blood mononuclear cells(PBMCs), which may vary between patients or even within one patient overtime. For example, this was accomplished by passing each sample through3 microfilters in series, and performing qPCR-TRAP on lysates from eachmicrofilter. As expected (FIG. 8B), serial filtration of 7.5 ml of bloodspiked with 20 cancer cells yielded high telomerase activity from thefirst filter (Ct=29.5, representing captured CTCs), and significantlylower activity from the 2nd and 3rd filters (33.5 and 33.4,respectively, representing background PBMCs). This approach enablestelomerase activity readings to be internally controlled for PBMCbackground.

FIG. 8A-8C show the measured telomerase activity of both recoveredcancer cells and control cells. Different Ct values (threshold cycle)mean the telomerase activity varied among different cells. Highertelomerase activity (lower Ct) is associated with a more aggressive,metastatic cancer phenotype. For example. PC3 cancer cells were capturedfrom whole blood on microfilter, localized by immunofluorescent staining(PE-conjugated anti-CD49 antibody), and recovered individually using amicropipette mounted onto a XYZ manipulating stage (FIG. 8C). Singlecancer cells were deposited in CHAPS lysis buffer and subjected toqPCR-TRAP, which yielded a significantly elevated telomerase activitylevel relative to negative controls. Slot microfilter live-capture canthus be used to assay telomerase activity or other biological orenzymatic processes from viable single CTCs (FIG. 8D).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Material and Methods

Cell Culture:

PC3 and DU145 cancer cell lines were maintained in RPMI/10% FBS at 37°C.

Example 1 Production of Parylene-C Slot Microfilter System FilterFabrication

As shown in FIG. 1, a parylene-C membrane filter was prepared accordingto the following processes.

-   -   1. Prime silicon wafer as substrate.    -   2. Coating of 10 μm parylene-C.    -   3. Ebeam deposition of 10 nm Cr and 200 nm Au.    -   4. Spin-coating of AZ1518 photoresist and lithography to form        pattern.    -   5. Wet-etching of metal layers using photoresist as mask.    -   6. Reactive Ion Etching (RIE) of parylene-C to form slots (an        array of 30,401 slots), using        -   metal as mask.    -   7. Strip off metal mask.    -   8. Parylene-C membrane is peeled off from silicon substrate.

Constant Pressure Fluid Delivery System

Pressure from a nitrogen tank was reduced below 1 psi by a two-stageregulator (FIG. 2) and further down regulated accurately by adjusting aneedle valve to 0.1-0.13 psi. A 15 ml conical tube containing the samplewas connected as a reservoir. The filter was sandwiched between two thinpieces of polydimethylsiloxane (PDMS) with wells to form a chamber,which was then clamped between PDMS/acrylic jigs from top and bottom toform a sealed system (FIG. 2).

Example 2 Use of Parylene-C Slot Microfilter-Based Enrichment of BloodCTCs Capture Efficiency, Cell Viability and Enrichment:

PC3 and DU145 human prostate cancer cell lines were stained withCalcein-AM fluorescent dye, and 10 cells were spiked into 1 ml humanblood obtained from healthy volunteers. After filtration, captured cellswere co-stained with Propidium iodide (PI) on filter and counted under afluorescent microscope (Zeiss Imager.Z1 microscope) with Axiovisionsoftware. Viable Calcein-AM-retaining cells were fluorescent green whiledead cells were fluorescent red by PI.

Capture efficiency was calculated as:

${{Capture}\mspace{14mu} {efficiency}\mspace{14mu} (\%)} = {\frac{{cancer}\mspace{14mu} {cells}\mspace{14mu} \left( {{green} + {red}} \right)\mspace{14mu} {on}\mspace{14mu} {filter}}{{cancer}\mspace{14mu} {cells}\mspace{14mu} {spiked}\mspace{14mu} {into}\mspace{14mu} {blood}} \times 100}$

Cell viability was calculated as:

${{Cell}\mspace{14mu} {viability}\mspace{14mu} (\%)} = {\frac{{green}\mspace{14mu} {fluorescent}\mspace{14mu} {cells}}{{total}\mspace{14mu} {captured}\mspace{14mu} {cancer}\mspace{14mu} {cells}\mspace{14mu} \left( {{green} + {red}} \right)} \times 100}$

Enrichment was determined by staining and counting the PBMCs remainingon filter with Acridine Orange and was calculated as:

${{Enrichment}\mspace{14mu} ({fold})} = \frac{\left( {{cancer}\mspace{14mu} {{cells}/{PBMCs}}} \right)_{{{on}\mspace{14mu} {filter}\mspace{14mu} {post}} - {filtration}}}{\left( {{cancer}\mspace{14mu} {{cells}/{PBMCs}}} \right)_{{original}\mspace{14mu} {blood}\mspace{14mu} {sample}}}$

7.5 ml blood samples were processed similarly but with the addition ofFicollpaque gradient centrifugation at 900 g for 30 min and resuspensionin 2 ml of PBS.

Patient Specimen Collection and Processing:

7.5 ml blood samples were drawn from patients with metastatic prostatecancer under an IRB-approved protocol, as well as from healthy volunteercontrols. All specimens were collected into EDTA K2 vacutainer tubes andprocessed within 24 hours of collection

Sample Processing

Blood Samples were processed as follows:

1. 7.5 ml blood is drawn from patient into ethylenediaminetetraaceticacid EDTA (K2) tube (BD #366643).2. Dilute the blood with 7.5 ml 1×PBS solution in a 50 ml conical tube.3. Add 4 ml of Ficoll-Paque (GE healthcare) into 15 ml conical tubes.Carefully layer 7.5 ml of the above diluted blood sample on top of theFicoll-Paque. For each sample, two 15 ml tubes are used.

4. Centrifuge at 900 g, for 30 min at 18° C.

5. Carefully transfer the PBMCs from the interface into 50 ml tubes.Combine the cells from the two 15 ml tubes for the same sample.6. Add PBS up to 50 ml and centrifuge at 900 g for 10 min.7. Resuspend the cell pellet in 1 ml PBS and transfer into 15 ml tube.8. Pass the sample through filter at 0.1-0.2 psi.9. Take out the filter and put into a 1.5 ml Eppendorf tube. Add 50 μlCHAPS lysis buffer (Millipore, Temecula, Calif.) on top of the filterand keep on ice for at least 30 min.10. Remove the filter and centrifuge at 14,000 rpm for 20 min at 4° C.Run qPCR telomerase activity assay as described below.

Scanning Electron Microscopy (SEM)

The microfilter containing captured cells was fixed, rinsed, and mountedper standard protocol, then photographed on a scanning electronmicroscope with 3,500× magnification (JEOL JSM/6390LV).

Example 3 Quantitative PCR (QPCR) Telomerase Activity Assay TelomericRepeat Amplification Protocol (TRAP) Assay

Telomerase activity from cell extracts was analyzed using a previouslydescribed real-time PCR-based telomeric repeat amplification protocol(TRAP) (Xu T, Xu Y, Liao C P, Lau R, Goldkorn A. Reprogramming murinetelomerase rapidly inhibits the growth of mouse cancer cells in vitroand in vivo. Mol Cancer Ther 2010; 9:438-49). The microfilter withcaptured cells was lysed in TRAPeze® 1×CHAPS Lysis Buffer (Millipore, 5Temecula, Calif.), and 5 μl of cell lysate per reaction was added foreach sample. For each reaction, DU145 cell lysates were used as standardcontrols in parallel.

QPCR telomerase activity assay was carried out as described below.

1. TS oligonucleotides (5′ AATCCGTCGAGCAGAGTT, 8 ng/μl) and 50 μM ofeach dNTPs are added to the cell lysate and incubated on a Bio-Rad MyiQThermocycler system for 30 min at 30° C. followed by inactivation oftelomerase at 95° C. for 1 min.2. ACX reverse primer (5′GCGCGGCTTACCCTTACCCTTACCCTAACC, 4 ng/μl), SYBRgreen (1:20,000) and 0.8U Taq platinum polymerase (Invitrogen, Carlsbad,Calif.) are added to the same tube in a master mix formula.

The reaction is run on the same machine for 40 cycles at 95° C., 0″; 50°C., 5″; 72° C., 10″, and the iQ5 optical system software version 2.0 wasused to analyze the results.

Example 4

FIG. 9 shows measurement of PSA transcript levels from filter-capturedcells. 100 LnCap cells were seeded into 1 ml fresh blood from healthydonor and passed through the parylene microfilter. RNA was isolated frommicrofilter-captured cells and cDNA was synthesized using commerciallyavailable kits. Both quantitative/real-time PCR (A) and standard PCR (B)were performed for PSA transcript detection and measurement. Compared tocontrol blood without seeded LnCap cells, blood with seeded LnCap cellshad least 12 fold PSA expression. These results constituteproof-of-principle for cancer cell capture by parylene microfilterfollowed by RT-PCR for detection and measurement of gene transcripts ofinterest.

What is claimed is:
 1. A method for diagnosing cancer or predicting adisease outcome in a patient sample, said method comprising: isolating acirculating tumor cell (CTC) from a sample using a parylene microfilterdevice comprising a membrane filter consisting of a parylene substratehaving an array of holes with a predetermined shape and size; anddetermining the telomerase activity of the isolated circulating tumorcell using a telomerase activity assay.
 2. The method of claim 1,further comprising correlating the telomerase activity with malignant ormetastatic potential.
 3. The method of claim 1, wherein the step ofdetermining the telomerase activity comprises: obtaining a telomeraseextension product; and amplifying the extension product by aquantitative PCR-based telomerase repeat amplification protocol assay.4. The method of claim 3, wherein the step of obtaining comprises: (i)lysing the isolated circulating tumor cell to produce a cell lysate; and(ii) mixing the cell lysate with an oligonucleotide that is a substratefor telomerase extension to obtain an extension product.
 5. The methodof claim 1, wherein the sample is a peripheral blood sample from apatient.
 6. The method of claim 1, wherein a quantitative PCR is used indetermining the telomerase activity of the isolated circulating tumorcell.
 7. The method of claim 1, wherein the step of isolating comprisingpassing the sample through the parylene microfilter device under aconstant pressure.
 8. The method of claim 7, wherein the pressure isbetween 0.1 to 0.3 psi.
 9. The method of claim 1, wherein the array ofholes is between 1 and 40,000 per square millimeter.
 10. The method ofclaim 9, wherein the array of holes is at least 5,000 per squaremillimeter.
 11. The method of claim 1, wherein the holes on the membranefilter have a shape selected from the group consisting of a circular, anelliptical, a rectangular, a symmetrical polygonal, an unsymmetricalpolygonal, an irregular shape and a combination thereof.
 12. The methodof claim 11, wherein the holes on the membrane filter have a rectangularshape.
 13. The method of claim 12, wherein the array of holes is between1 and 850 per square millimeter.
 14. The method of claim 12, wherein theholes have a dimension of 5.5 μm to 6 μm by 40 μm.
 15. The method ofclaim 1, wherein the membrane filter has a hole dimension ranging fromabout 5 μm to about 12 μm.
 16. The method of claim 1, wherein themembrane filter has a figure of merit up to 890%/hr.
 17. The method ofclaim 1, wherein the thickness of the membrane is at least about 1 μm.18. A method for detecting and quantifying the telomerase activity of acirculating tumor cell, said method comprising: isolating thecirculating tumor cell (CTC) from a sample using a parylene microfilterdevice comprising a membrane filter consisting of a parylene substratehaving an array of holes; and determining the telomerase activity of theisolated circulating tumor cell using a quantitative PCR telomeraseactivity assay.
 19. The method of claim 18, wherein the step ofdetermining comprises: obtaining a telomerase extension product; andamplifying the extension product by a quantitative PCR-based telomeraserepeat amplification protocol assay.
 20. The method of claim 19, whereinthe step of obtaining comprises: (i) lysing the isolated circulatingtumor cell to produce a cell lysate; and (ii) mixing the cell lysatewith an oligonucleotide that is a substrate for telomerase extension toobtain an extension product.
 21. The method of claim 18, wherein thesample is a peripheral blood sample.
 22. The method of claim 18, whereina quantitative PCR is used in determining the telomerase activity of theisolated circulating tumor cell.
 23. The method of claim 18, wherein thestep of isolating comprises passing the sample through the parylenemicrofilter device under a constant pressure.
 24. The method of claim23, wherein the pressure is between 0.1 to 0.3 psi.
 25. The method ofclaim 18, wherein the array of holes is between 1 and 40,000 per squaremillimeter.
 26. The method of claim 25, wherein the array of holes is atleast 5,000 per square millimeter.
 27. The method of claim 18, whereinthe holes on the membrane filter have a shape selected from the groupconsisting of a circular, an elliptical, a rectangular, a symmetricalpolygonal, an unsymmetrical polygonal, an irregular shape andcombinations thereof.
 28. The method of claim 27, wherein the holes onthe membrane filter have a rectangular shape.
 29. The method of claim28, wherein the array of holes is between 1 and 850 per squaremillimeter.
 30. The method of claim 28, wherein the holes have adimension of 5.5×40 μm.
 31. The method of claim 18, wherein the membranefilter has a hole dimension ranging from about 5 μm to about 12 μm. 32.The method of claim 18, wherein the membrane filter has a figure ofmerit up to 890%/hr.
 33. The method of claim 18, wherein the thicknessof the membrane is at least about 1 μm.
 34. A method for enrichment ofcirculating tumor cells, said method comprising: passing a samplecontaining a circulating tumor cell through a parylene microfilterdevice comprising a membrane filter consisting of a parylene substratehaving an array of holes with a predetermined shape and dimension; andcapturing said circulating tumor cell on the membrane filter, whereinthe enrichment of circulating tumor cells is greater than 200-fold. 35.The method of claim 34, wherein said passing is under a constantpressure.
 36. The method of claim 35, wherein said capturing has greaterthan 90% capture efficiency.
 37. The method of claim 36, wherein thecirculating tumor cell has greater than 90% cell viability.
 38. A systemfor isolating a circulating tumor cell, said system comprising: aparylene microfilter device comprising a membrane filter consisting of aparylene substrate having an array of holes with a predetermined shapeand dimension; and a constant pressure delivery system coupled to theparylene microfilter device for maintaining a constant pressure.
 39. Thesystem of claim 38, wherein the pressure is between 0.01 to 0.3 psi. 40.The system of claim 38, wherein said array of holes has a rectangularshape.
 41. A method for measuring mRNA expression levels of a gene in acirculating tumor cell (CTC), said method comprising: isolating a CTCfrom a sample using a parylene microfilter device comprising a membranefilter consisting of a parylene substrate having an array of holes witha predetermined shape and size; and measuring the mRNA expression levelsof the gene in said CTC.
 42. A method for staining a circulating tumorcell (CTC), said method comprising: isolating a CTC from a sample usinga parylene microfilter device comprising a membrane filter consisting ofa parylene substrate having an array of holes with a predetermined shapeand size; and staining said CTC with a dye or an antibody conjugated toa dye.
 43. A method for propagating a circulating tumor cell (CTC) inculture, said method comprising: isolating a CTC from a sample using aparylene microfilter device comprising a membrane filter consisting of aparylene substrate having an array of holes with a predetermined shapeand size; and propagating said CTC in culture.
 44. A method formeasuring telomerase activity in a single circulating tumor cell (CTC),said method comprising: isolating a group of CTCs from a sample using aparylene microfilter device comprising a membrane filter consisting of aparylene substrate having an array of holes with a predetermined shapeand size; obtaining individual CTCs from the group of CTCs isolated fromthe sample; and determining telomerase activity of said CTC.
 45. Themethod of claim 44, wherein the step of obtaining comprises: using amicropipette to recover individual CTCs from the group of CTCs isolatedfrom the sample.